Università degli studi di Sassari Scuola di Dottorato di Ricerca Scienze dei Sistemi Agrari e Forestali
e delle Produzioni Alimentari
Indirizzo Scienze e Tecnologie Zootecniche Cycle XXIV
Approaches for the analysis of genetic diversity in cattle
breeds farmed in Italy
Dr.: Elia Pintus
Direttore della Scuola: prof. Giuseppe Pulina Referente di Indirizzo: prof. Nicolò P.P. Macciotta Docente Guida: prof. Nicolò P.P. Macciotta Tutor: Dott.ssa Silvia Sorbolini
Index Chapter 1 - General Introduction
1. The three levels of biodiversity Pag. 1
2. The resource biodiversity Pag. 4
3. The birth of agriculture Pag. 5
4. The phenomenon of cattle breeds Pag. 7
5. The principles of genetic selection of livestock species
Pag. 8
6. The process of extinction Pag. 10
7. The breeds and molecular genetics Pag. 11
8. Molecular traceability Pag. 14
9. The identification of selection signatures Pag. 17
Objectives of the thesis Pag. 19
References Pag. 20
Chapter 2 - Melanocortin 1 receptor (MC1R) gene polymorphisms in three Italian cattle
breeds
1. The case of the Sardo-Modicana breed Pag. 27
2. Principles of product traceaility Pag. 30
3. The pigmentation in mammals Pag. 30
References Pag. 36
Abstract Pag. 42
Introduction Pag. 44
Materials and methods Pag. 47
Results Pag. 50
Discussion Pag. 53
Acknowledgements Pag. 56
Chapter 3 - Use of Locally Weighted Scatterplot Smoothing (LOWESS) regression to
study genome signatures in Piedmontese and Italian Brown cattle breeds
1. Selection signatures Pag. 61
References Pag. 64
Summary Pag. 66
Introduction Pag. 67
Materials and methods Pag. 70
Results and discussion Pag. 74
Conclusion Pag. 84
Acknowledgments Pag. 84
References Pag. 85
Chapter 4 - General Conclusions Pag. 91
CHAPTER 1
General Introduction
The exact definition of the term “biodiversity”, was coined in 1988 by E.O. Wilson with the aim of replacing the term “biological diversity” which was considered less efficient in terms of communication. This definition opened, in the international scientific community, a debate that has not come yet to an end. Omitting definitions outdated or overly philosophical and eccentric, the most used definition is the one written in 1992, “Biodiversity is the variety of ecosystems that include both communities of living organisms in their particular habitats, both the physical conditions under which they live”.
Therefore biodiversity must be interpreted as diversity within species, between species and between ecosystems. Subsequent and different elaborations of the same concept have led to the definition adopted by the United Nations Convention on Biological Diversity of Rio de Janeiro: biodiversity is “the variability among all living organisms including, the subsoil, of air, aquatic and terrestrial ecosystems, marine and ecological complexes of which they are part” (UNEP, 1992).
1. The three levels of biodiversity
Biodiversity is, as already mentioned, the variety with which all the living parts of a place or territory occur, and it is the term commonly applied to different levels of biological organization (Harper and Hawksworth, 1995). Within species, individuals are all different from each other because of differences at DNA level and, therefore, genetic. It is now possible, by the use of
genetic techniques to quantify the diversity at this level and then talk about
genetic diversity.
Genetic diversity refers to the presence of different forms of genes in the genetic material of a single species (Templeton, 1995). In almost all multicellular organisms, the genetic information of an individual is not identical to the one of other individuals, because each of them represents a unique combination of genes within a species. This is a consequence of: sexual reproduction, genes recombination and spontaneous mutations induced in the structure of the genes.
The environment, with all its different aspects, acts on individuals determining death or survival. The final result is, therefore, a selection of various and possible combinations of genes (Falconer and Mackay, 1996). This is the reason why two isolated populations, even if they belong to the same species, may undergo a different selection due to the action of various environmental factors that, in the long run, can bring the two populations to have two distinct gene pools. This phenomenon can occur in relatively confined spaces and it is extremely important because it contributes to the creation of genetic diversity for determining the adaptability of the species during evolution. A population or a specie, which lost part of its gene pool, and then lack of genetic variability, is in danger of extinction because lose part of its potential adaptability to new and different environmental conditions (Colwell, 2009). Moreover, the loss of gene pool may lead to an increase of the frequency of unfavorable genes resulting in a further increase of the risk of extinction. All genes distributed in the totality of living beings in the world do not contribute equally to the global genetic
diversity. Genes that regulate fundamental biological processes are preserved unchanged under the different groups of species (taxa) and, generally, exhibit a lower degree of variation. More specialized genes show a larger range of variability.
It is possible to distinguish the different species that populate a certain environment. This level of diversity is species diversity and refers to the variety of species that live in close contact in a specific environment. Its aspects can be analyzed and studied in different ways. However the most popular types of measurement are:
− species richness; − abundance of species;
− phylogenetic or taxonomic diversity.
The number of species is commonly defined as species richness and is one of the possible measures of the biodiversity of a specific environment. It can be used as a basis for comparison between different places. The species richness is considered the simplest measure of biodiversity, it is quite easy to evaluate (Christie et al., 2004). However, it is incomplete and it gives an approximation of the variability present among the living beings.
The estimate of abundance of species, evaluates the abundance of single species within the community. Changes of abundance of species is another aspect of diversity and it is measured with a standardized index on a scale ranging from values close to 0, indicating low uniformity or domination of a single species, to 1 that indicates the maximum homogeneity between species (Stirling and Wilsey,
2001). Another approach to measure species diversity is to consider the phylogenetic and taxonomic diversity. It is based on the study of genetic relationships between different groups of species (Faith, 1992). This type of measure leads to a hierarchical classification represented by a dendrogram whose branches represent the phylogenetic evolution of the taxa examined (Faith and Baker, 2006).
Measurements within the species are usually considered the most suitable to analyze the diversity between the organisms, because the species are the primary goal of the evolution and are relatively well defined.
Biodiversity is also defined as a measure of the complexity of an ecosystem and of the relationships between its components. The analysis of the availability of different ecosystems in a particular environment or in a distinct geographic area, is the analysis of diversity of ecosystem. The assessment of the ecosystem diversity has critical points due to the complexity of finding the limits of the ecosystem (Christie et al., 2004). The classification of the immense variety of all ecosystems on Earth remains one of a major goal of science and it is important for the management and conservation of the biosphere.
The importance of protecting ecosystems to preserve nature and species, within Community rules, has been recognized with the Habitats Directive (92/43/EEC).
2. The resource biodiversity
Throughout its history man has gradually created a niche that, especially in urban areas, has excluded him from contact with the natural environment. Also
the continuous advancement of new technologies for the industrial exploitation of natural resources have made humanity able to radically change the appearance and the balance of the natural environment. Biodiversity is essential for humans because it yields the nutrients, oxygen for respiration, medicines, natural fibres for textiles, raw materials for the production of energy and even the processes of purification and recycling of waste products.
Therefore, the loss and the reduction of biodiversity not only changes the ecosystem functions essential for life but it also has negative economic impacts represented by reduced food, energy and genetic resources. Although the study of multiple forms of life on earth has a very far roots, it now represents a crucial tool to urgently address the problem of loss of biodiversity.
3. The birth of agriculture
The history of agriculture began about 13,000 years ago. In this period began the first attempts at domestication of the main species of livestock and crop plants. This process has inevitably led the man to have a high ability to control food productions. The main consequence has been the occurrence of major demographic and technological changes. The domestication of animals is still considered one of the most important moments of the history and, most likely, the spark that led to an initial growth of human civilizations (Diamond, 2002).
Thousands of years of evolution and selection have contributed to the growth of diversity (Groeneveld et al., 2010), creating the conditions to practice the farming of the species in different environment conditions. Diversity is
essential for all production systems, because it provides the raw material for the improvement of breeds and to adapt to changing conditions.
However, only a little part of the total species present on earth have been completely domesticated. In fact, the process of domestication has been extremely complex and gradual (FAO, 2007). Causes that first led man to domesticate animals remain a mystery and almost certainly they may be different from one geographical area to another or from one species to another. The tendency of the human to groped to tame wild animals is the basis of the domestication (Diamond, 2002). The great expansion of human populations, mainly due to climatic changes, probably represents the main cause that has led to the domestication of animals. Another cause is represented by the increased requirement of food. Finally, the same amount of calories of food energy could be produced by using less energy by means of agricultural practices rather than by hunting and gathering (Gupta, 2004).
Today some wild ancestor (i.e. auroc) and many breeds of farmed species are extinct or highly endangered with extinction (Taberlet et al., 2008). For these species, domestic animals are now a sort of biological bank that inherited diversity from their wild ancestors. Unfortunately most of this genetic diversity has been lost nowadays.
As already mentioned, only a small part of total animal species has been successfully domesticated. The explanation can be found in the characteristics or advantages required by the domestication itself (FAO, 2007). In fact, from the beginning of this phenomenon, some characteristics were more important:
adaptability; rapid growth rate; short intervals between births and offspring of large size (Diamond, 2002). Most of the ancestral species have been identified and through molecular studies was obtained a reconstruction of the history of breeds and ancestral populations (Groeneveld et al., 2010).
4. The phenomenon of cattle breeds
Since Neolithic age, cattle have spread all over the world following the migrations of human populations or because of trade. Once that new territories were reached, cattle gradually adapted to specific environmental conditions and was farmed in the new area. It was only 200 years ago that these differences between animals of the same species were defined and the concept of breed was introduced (Ajmone-Marsan and The GLOBALDIV Consortium, 2010).
After the industrial revolution, some of the traditional livestock productions lost their importance due to the availability of new industrial products. On the other hand, the demand of proteins of animal origin was continuously increasing. Therefore, an intense selection of breeds of livestock for food production started. Since then, specialized breeds and intensive production systems have spread around the world.
On the contrary, autochtonous populations not subjected to any selective pressure, have survived in areas where intensive farming had not been able to affirm due to economic, cultural or environmental conditions. Thus local or native breeds are now generally characterized by their limited geographical distribution (Hiemstra et al., 2010).
− present in a single country; − cosmopolitan.
The former breeds are commonly referred to as “local”, whereas the latter as a “transboundary”. In agreement with FAO (2007) the transboundary breeds, can be subdivised in: breed present in more than one country but within a single region (regional transboundary), and breed present in all countries and more than one region (international transboundary).
5. The principles of genetic selection of livestock species
Genetic improvement is the process of modification of genetic heritage in order to improve the characteristics in the farmed species. This process has been often done, especially in the past, in unconscious and empirical way through the selection of phenotypes that were considered more favorable. Currently, thanks to modern techniques, this process is a combination of phenotypic observations with genotypic knowledge available from genome studies.
The results obtained in the field of genetic improvement in the millennia, since the domestication in the Neolithic period, are small compared to those obtained from the early years of the last century. In fact it is from the beginning of the twentieth century that the selection underwent to a revolution, largely due to the development of technical factors and scientific achievements that have made it a continuously evolving process. Today the main objective of livestock breeding is to be able to estimate with great accuracy the genetic merit of the individual. One of the first attempts to estimate the genetic value of selection candidates was the Selection Index (Hazel, 1943). According to this approach,
the breeding value for a quantitative trait is estimated using the phenotypes previously adjusted for some fixed effects. However, this method has some problems. It does not take into account genetic differences between generations or farms. For this reason, reliable results can be obtained only for animals farmed in the same environmental conditions.
The first applications of the BLUP (Best Linear Unbiased Prediction) method allowed to estimate simultaneously fixed effects and random additive genetic effects of the bulls (Henderson, 1975). This methodology has been used in genetic evaluation systems of many countries. However, early BLUP models considered only the male population (i.e. Sire and Maternal Grandsire models). Thus the estimated breeding value was only half of daughters additive genetic effect because only fathers were evaluated. With the Animal model, geneticists able to estimate the genetic merit of all animals within a breed. However, due to the large number of equations in the model, the routinely use of this approach had been feasible only when adequate computer resources were available.
The above mentioned methods take into account the total production per lactation of standardized length. The cumulated yields were obtained from Test Day (TD) data recorded on farm. The main limitation of the so called lactation models is that they are not able to take properly account of environmental effects (i.e. climate and feeding) that may affect specifically production in some lactation stages.
The Test Day Model (Stanton, 1992) provides the solution to this problem through the direct analysis of data obtained from daily production. Generally
these models require high computational resources. Moreover, they are very sensitive to the precision of the phenotypic data. Incorrect production, because obtained from imprecise controls, produce unreliableindices.
6. The process of extinction
Extinction is a natural process which is considerably accelerated by human activities (Martens et al., 2003). In general, the phenomenon involves all flora and fauna species. Recent studies reported that about 20% of all bovine breeds of the world resulted to be at risk. Actually they have a number of females less than or equal to 1,000 and about 9% of them are already extinct (FAO, 2007).
According to the information collected in European and worldwide databases (EFABIS (http://efabis.tzv.fal.de/) and DAD-IS (www.fao.org/dad-is/)), the local European cattle breeds present data even more alarming, with about 40-50% of them that be considered at risk and some other are actually extinct (www.fao.org/DAD-IS). For this reason, the majority of European cattle breeds can be classified as local breeds. Agriculture public organizations are increasingly oriented to understand the state of European local populations in order to develop best policies and strategies for the conservation and the maintenance of genetic diversity of cattle in Europe (Hiemstra et al., 2010).
The aim of conservation is to preserve breeds and agricultural production systems able to satisfy the maintenance of genetic variability (Negrini et al., 2006) and of cultural, social, economic and environmental values. From a genetic point of view, the importance of diversity safeguarding between and within the
breed is widely recognized. For all this reasons it is important to determine risk status or state of damage of a breed which is commonly estimated based on the number of animals. The level of risk is difficult to assess accurately. For this reason it is important to examine demographic and genetic factors, that have been defined as a probable indicators of a future extinction of a breed (Gandini et al., 2004).
7. The breeds and molecular genetics
Several investigations with molecular tools have been carried out on European local cattle breed to study their origin and genetic differentiation.
Archaeological findings indicates that the cattle entered in Europe through two main roads: the way of the Danube through the lowlands of Central Europe and the way along the Mediterranean coast (Pinhasi et al., 2005). Further molecular studies (Negrini et al, 2007) found that two main groups of cattle breeds can be distinguished in Europe:
− podolica, as many Italian and Hungarian breeds; − other cattle breeds.
Molecular analysis are not only used for evolutionary studies but are now also used to measure the differences between or within breeds. The neutral markers reflect the overall genomic change and are able to highlight differences in breeds and the potential variation in traits not yet subjected to selection. The first research applications concerning genetic markers in livestock animals were made using biochemical and immunological markers. But it was with the use and
development of technologies for DNA markers that significant advancements in the knowledge of the structure of the genome of livestock species were achieved.
The first used DNA markers were Restriction Fragment Length Polymorphisms (RFLPs) (Kan and Dozy, 1978). These markers are very frequent in the genome and give the possibility to build genetic maps in species of zootechnical interest (Beckmann and Soller, 1983). Moreover, they allow for the identification of loci responsible for quantitative genetic variation (quantitative trait loci: QTL). However, RFLPs had a little application due to the identification method based on the technique of Southerm blotting which is long and laborious. Another reason is that, in general, RFLPs have only two alleles. Another type of markers, subsequently identified, were the minisatellites or Variable Number of Tandem Repeats (VNTRs) (Nakamura et al., 1987), which have the same problems of RFLP about laboratory analysis, but have the advantage to have high number of alleles.
RFLPs and VNTRs were used in the first phases of the construction of genetic maps. Currently they have been replaced by other markers such as microsatellites, which can be easily analyzed using the PCR technique. The development of microsatellites has allowed remarkable progress in the analysis of the genome. These markers are characterized by a variable number repetitions sequences of 1-5 nucleotides and are highly informative thanks to their high number of alleles (Litt and Luty, 1989; Weber and May, 1989). In general, microsatellites are found in anonymous DNA regions, i.e. regions without known function. The use of automated sequencers for their analysis and use of software
for data preservation and interpretation, has contributed to make microsatellite the most used markers for the genetic maps construction and QTL analysis. Other types of markers are:
− RAPDs (Random Amplified Polymorphic DNA) (Williams et al., 1990; Welsh and McClelland, 1991) that identify markers through short oligonucleotides as primers in PCR;
− The AFLPs (Amplified Fragment Length Polymorphisms) (Vos et al., 1995) that combine the restriction analysis of DNA with PCR and allow, using different combinations of enzymes and primers, for the simultaneous analysis of a large number of loci.
Other methods allow to identify more efficiently polymorphisms caused by point mutations, such as Single Nucleotide Polymorphisms (SNPs). These markers are the most widespread in the animal genome (one every 500-3000 nucleotides). Among these, the Single Strand Conformation Polymorphisms (SSCP) method (Orita et al., 1989) allows the identification of point mutations in amplified DNA fragments of 100-400 nucleotides. More recently, have been developed further methods of analysis commonly known as high-throughput, which allow high efficiency and speed in typing of SNPs. Among these we can mention:
− methods based on minisequencing primer extension (Syvänen, 1999); − methods based on the chromatographic principles such as DHPLC
(Denaturing High Performance Liquid Chromatography) (Huber et al., 1993);
− methods that use the mass spectrometry techniques, such as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).
Additional methods are based on the use of solid supports (microarray) on which are fixed high-density oligonucleotides (Chee et al., 1996), which allow to simultaneously analyze hundreds of SNPs. All these technologies enable geneticists to analyze a large number of markers in a short time.
8. Molecular traceability
Today more than 40 animal species contribute to the production of food of animal origin. The combined selection pressure due to environmental factors and the controlled breeding imposed by humans, have led to the creation of large variety of genetically distinct breeds. The development of this diversity, which occurred over thousands of years, is a valuable resource for the breeding of livestock species. In fact, genetically different populations can positively deal problems such emerging threats, new human knowledge and nutritional requirements, fluctuating market conditions or, in general, changing societal needs (FAO, 2007).
It is clear the importance of biodiversity conservation and environmental protection, especially for biological areas particularly defined and limited. This phenomenon is of particular importance in the case of so-called minor livestock breeds, that are farmed in areas defined marginal and with which show a particular symbiosis. It is through the conservation, protection and rational
farming of these breeds that a sustainable economy can be created in areas that otherwise would face a gradual decline and abandonment (Davoli, 2011).
In Italy, 23 autochthonous cattle breeds have been recorded. They are distributed in highly fragmented and localized areas (Bigi and Zanon, 2009). To emphasize their great adaptability and strong ability to interact with the surrounding environment, these breeds are commonly defined as local breeds.
In general, they are characterized by:
− High capacity to adapt to the extreme environment conditions;
− ease of delivery, that is essential to ensure the survival of the calf in the wild farming;
− good maternal ability, remarkable ability to raise the calf in good food condition until weaning;
− high reproducibility, i.e. high sexual precocity, fertility and reproductive longevity;
− compatible with the farming environment, large size and bulk associated with strong skeletal framework.
Local breeds are farmed all over the world always in agronomically difficult areas that can not be used with specialized breeds or with an higher production performance.
A useful tool for the protection and enhancement of typical products that may lead to the development of marginal areas by encouraging the conservation of biodiversity and consequently the protection of local breeds is represented by molecular traceability (Crepaldi et al., 2008). It is defined as the ability to control
the origin of the products and the identity of the animals throughout the production chain through the use of technologies that allow direct analysis of DNA (McKean, 2001). Moreover, molecular traceability combined with a control system for food hygiene and safety, can protect consumers from fraud, help some categories as people suffering from food allergies or intolerances.
Also agriculture has suffered the effects of the markets globalization. Today most of the raw materials used for human nutrition are bought where they are cheaper, preventing the consumer to know the origin of food. Moreover, especially in Europe, in recent years, consumer confidence in food of animal origin declined significantly due to dioxin and BSE scandals.
All these facts has led to an ever-increasing attention of consumers to health and origin of food products. This represent a potential chance of development of marginal areas, typical productions and the consequent conservation of local breeds. The most actual example is represented by the growing interest in products marketed in areas very close to the place of production.
The traceability can be classified into (Crepaldi et al., 2008): − individual traceability;
− traceability of breed; − traceability of species.
The individual traceability allows to trace back a product to the individual it was obtained from. However, the implementation of this type of traceability is rather complex. A database of individual biological samples of farmed animals is
needed. In practice it is used only for those products that are really obtained from individual animals. The traceability of species can to attribute a product of animal origin to the species that produced it, and is useful in order to certify if a particular cheese was made using only milk from a particular species. The traceability of breed allows to assign a product, or the same animal, to a particular breed. This type of traceability has gained considerable importance due to the diffusion of so-called mono-breed products.
9. The identification of selection signatures
Molecular markers are used to study the genome at various levels and for different reasons. They are represented by locus-specific variations transmitted in Mendelian way from one generation to the next. Panels of high-density SNPs have made the use of markers a useful tool for identifying genome region affected by a selection (Colli et al., 2011).
Contrary to some evolutionary forces that act indiscriminately throughout the genome (Luikart et al., 2003), selection acts on specific points. It changes, for example, diversity within a breed or genetic distance between breeds that have been selected for different production attitudes.
Recent studies (Hayes et al., 2008; Prasad et al., 2008) investigated the difference in allele frequencies of breeds selected for different traits. Selection points in areas very close to genes that influence milk or meat production (i.e. STAT1, ABCG2, DGAT1 and TG) have been identified (Hayes et al., 2008).
Currently, different approaches and methods are used to identify signatures of selection (Biswas and Akey, 2006). Among these the fixation index
Fst, quantifies the level of differentiation between subpopulations (Weir and Cockerham, 1984) and one of his possible interpretations is the analysis of heterozygosity level between populations. Fst values higher than expected show a divergent selection, on the contrary lower values show an uniformity of selection. In general in the domestic breeds range of Fst values from 0,005 and 0.3, and values of 0.15 indicate significant differences between two populations (Frankham et al., 2002)
Additional approaches are: I) methods based on polymorphisms within species: Tajima’s D (Tajima, 1989); Fu and Li’s D and F (Fu and Li, 1993); Fay and Wu’s H test (Fay and Wu, 2000); Long range haplotype (LRH) test (Sabeti et al., 2002); iHS (Voight et al., 2006); LD decay (LDD) (Wang et al., 2006), II) tests based on polymorphisms within species and the divergence between species: Hudson–Kreitman–aguade (HKA) test (Hudson et al., 1987); McDonald Kreitman (MK) test (McDonald and Kreitman, 1991), III) tests between species: dn/ds test (Suzuki and Gojobori, 1999).
Objectives of the thesis
General aim of the research developed during my PhD was the study of genetic differences between cattle breeds farmed in Italy. This purpose has been pursued by addressing two different issues. The first was the use of a specific gene as marker for the traceability of products obtained by local breeds farmed in low input systems. The second was the study of selection signatures in two Italian cattle breeds with different breeding goals, dairy and beef, using data generated by a high throughput SNP platform and a specifically adapted statistical procedure.
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CHAPTER 2
Melanocortin 1 receptor (MC1R) gene polymorphisms in three Italian cattle breeds
1. The case of the Sardo-Modicana breed
In the Island of Sardinia there are three local cattle breeds that are characterized by peculiar reproductive and productive traits (Brandano et al., 1984). They are Sarda, Sardo-Bruna and Sardo Modicana. The existence of the Sarda breed is documented since the prenuragic age. It almost certainly derived from the western Mediterranean cattle breeds (especially Iberian) with possible influences of North African and Middle Eastern breeds. During its evolution, the Sarda has been affected, sometimes very markedly, by other breeds. In particular, Brown Swiss bulls were imported from Switzerland in the northern areas to improve milk and meat production of the Sarda. In the south part of the Island, where agriculture was more developed, Modicana bulls from Sicily were imported to improve size and strength of the local cattle for work purposes (Brandano et al., 1984). Previous studies carried out on somatic measurements and on blood and milk genetic markers highlighted that the Sarda is actually a very heterogeneous population rather than a well-defined breed. Animals show marked differences in general conformation, coat color (which varies from black to red and from uniform to bi-color) and size. The total number of animals has been estimated in about 16,700 in 2011 (AIA, 2011) (Table 1).
The Sarda can be found in the most inaccessible areas of the Island (Barbagia, Iglesiente, Sarrabus and Gallura). The farming system is almost exclusively extensive. The breed is characterized by a poor attitude to meat yield: very low average daily gain and dressing percentage. However it is characterized by a relevant fertility, calving ease and maternal attitude.
Table 1: number of farms and animals raised for Sarda breed (From: thesis of Angelo Zedda) Province Herds number Animals registered Animals not registered Undefined animals Total Ca 128 2,936 331 678 3,945 Nu 409 8,716 196 1,302 10,214 Or 25 168 6 101 275 SS 115 1,967 76 167 2,210 Tot. 677 13,787 609 2,248 16,644
For these reasons, the breed is mainly used to produce F1 crosses with specialized beef breeds in marginal areas that cannot be exploited by other animal farming systems (Brandano, 2008).
The Sardo-Modicana breed was obtained by cross of Sardinian hill-breed with bulls of Modicana breed from Sicily, imported from some local breeder around 1870 from the province of Ragusa. The aim of the crossbred was to improve the the size and strength of work of the animals. This was the main attitude of the breed until the spread of mechanization in agriculture. In the period of maximum diffusion (decade 1940-1950) the farming area covered the central (Montiferru, Planargia) and the southern part (Trexenta, Marmilla and Campidano) of the Island. After the massive introduction of mechanization in agriculture, the Sardo-Modicana breed lost its main productive function (Brandano et al., 1983) and a reduction of the number of animals started. Currently the Sardo-Modicana is farmed in the mountain areas of Montiferru and Planargia. The Sardo-Modicana is characterized by a robust skeleton, a red coat, medium size, high calving ease and good maternal ability. It is used, either pure-breed or in crosspure-breed with beef bulls, for meat production (Brandano, 2008). The milk that exceeds the amount suckled by the calf is used for the production
of the typical cheese “Casizolu”. The present size of the Sardo–Modicana population is about 3,400 animals (AIA, 2011) (Table 2).
Table 2: number of farms and animals raised for Sardo-Modicana breed (From: thesis of Angelo Zedda)
Province Herds
number registered Animals not registered Animals Undefined animals Total
Ca 29 384 85 50 519
Nu 19 149 1 12 162
Or 79 1,473 723 351 2,547
SS 19 142 6 13 161
Tot. 146 2,148 815 426 3,389
Also this breed is farmed extensively. In spite of the quality of its production, that are highly appreciated by consumers, the farming of this breed experiences a deep crisis. Apart from the overall problems of agriculture, the breed suffers from the specific issue of local population, i.e. the markedly lower production levels compared to specialized breeds.
A strategy for the valorization of the Sardo-Modicana breed can be found in the genetic characterization and the development of methods for products identification and traceability. A successful example of genetic traceability for typical products in cattle breeds is represented by the MC1R gene polymorphism cattle breeds (Kantanen et al., 2000; Rouzard et al., 2000; Graphodatskaya et al., 2002; Maudet and Taberlet, 2002; Gan et al., 2007; Mohanty et al., 2008). Several authors have suggested that the MC1R gene alleles can be used as breed-specific markers for animal products traceability (Maudet and Taberlet, 2002; Crepaldi et al., 2003; Rolando and Di Stasio, 2006). In addition, the MC1R gene polymorphism has recently been analyzed in some Italian cattle breeds. In fact, in
Italy this gene has been used to distinguish the Parmigiano Reggiano cheese made exclusively with milk of Reggiana breed, from cheeses obtained from other breeds such as Holstein Friesian and Italian Brown (Russo et al., 2007). For this reason it can be used as a specific marker for the traceability of products obtained from local breeds such Sardo-Modicana.
2. Principles of product traceability
The assignment of a subject to a breed by using molecular methods can be carried out through two strategies:
1. the probabilistic approach; 2. the deterministic approach.
The first provides the creation, for each genotyped breed whit highly polymorphic markers, a database with information on the alleles present and their frequency. The individual to be assigned is analyzed with the markers mentioned above and the assignment is made probabilistically, starting from allele frequencies of each breed or from genetic distances between breeds.
The deterministic approach involves the search of specific molecular markers of a breed and/or of genes with specific allelic variants. The genotyping of these markers would allow to assign an animal directly to a specific breed without the need to carry out any probabilistic calculation (Mariani et al., 2005).
3. The pigmentation in mammals
The pigmentation in mammals is based on the presence or absence of the melanin in hair and skin. Melanins are formed by enzymatic oxidation of amino acid tyrosine. Two types of pigments are derived:
− Eumelanins; − Pheomelanins.
The pigmentation is determined by the distribution of Eumelanins and Pheomelanins which are responsible of a black/brown and yellow/red colors, respectively (Prota, 1992; Nordlund et al., 1998).
The metabolic pathways that lead to the synthesis of these two types of melanin are largely unknown. The key enzyme is the tyrosinase, which catalyzes the metabolic steps that start from tyrosine idroxylation and leads to the synthesis of dopaquinone that is a common precursor of these two types of melanin. In absence of thiol compounds undergoes intermolecular cyclization leading to the production of eumelanin. In presence of thiols it gives rise thiol adducts of Dopa termed cysteinyldopas and leads to pheomelanin production (Figure 1) (Lamoreux et al., 2001).
The processes of synthesis and accumulation of melanin occur in melanosomes, which are specific cytoplasmatic organelles of specialized cells called melanocytes, which reside between dermis and epidermis. Subsequently, the melanosomes are transferred in the hairs during their growth through a exocytosis process. The migration of melanocytes occurs during embryo development. They start from the neural crest and move in different parts of the body conferring the pigmentation to the areas where they operate.
Moreover, in some parts of the body the same pigmentation can be changed depending on the level of activity of melanocytes (Seo et al., 2007). First studies on the genetics of coat color were made at the beginning of 1900
(Barrington and Pearson, 1906) just after the rediscovery of Mendel’s law. These researches were followed by other studies on the pigmentation similarities between different mammals.
Figure 1: Metabolic pathways that lead to the synthesis of two types of melanin (From: Lamoreux et al., 2001)
The analyses of segregation of colors allowed for the identification of key genes that affect coat color in mammals (Searle, 1968; Olson, 1999). Thanks to the knowledge derived from embryology, biochemistry and molecular genetics has been possible to define the functions of these genes. According to Russo and Fontanesi (2004) they can be classified as follows:
Genes involved in the regulation of melanogenesis:
− the Agouti locus (A) that encodes a protein (agouti signaling protein, ASIP), which acts as an antagonist of α-melanocyte-stimulanting hormone (α-MSH) in the MC1R receptor.
The E and A locus show epistatic effects. In various mammalian species, dominant alleles at the locus E produce a black coat color, whereas recessive alleles produce to a red/yellow color. Alleles at the locus A cause the recessive black color only when at the locus E the wild-type allele is present, but not dominant or recessive allele (Russo and Fontanesi, 2004).
Genes that influence the development of melanocytes and their migration during embryogenesis:
− the locus White Spotting (W), identified at molecular level in KIT gene; − the locus Roan (R) coding for mast cell growth factor (MGF) that binds to
the KIT gene.
Genes that encode enzymes for the biosynthesis of melanin: − the Albino locus (C) coding for the enzyme tyrosinase (TYR);
− the Brown locus that encodes for the enzyme tyrosinase-related protein 1 (TYRP1);
− the Slaty locus that encodes for the enzyme tyrosinase-related protein 2 (TYRP2).
Genes that influence the morphology of melanocytes:
− the locus Dilute (D), which encodes for a type V myosin (MYO5A). Genes that influence the structure and function of melanosomes: − Locus Silver (PMEL17);
− pink eyed dilution locus (p) that encode for a melanosomes transmembrane proteins.
The Extension locus was initially characterized at the molecular level in mice (Robbins et al., 1993). This locus encodes for melanocortin receptor 1 (MC1R) also referred to as melanocyte stimulating hormone receptor. The MC1R is a transmembrane protein of Gprotein-coupled receptors family (Robbins et al., 1993). As well as in mice, in humans (Valverde et al., 1995), horse (Marklund et al., 1996), sheep (Vage et al., 1999), chicken (Takeuchi et al., 1997) and in pig (Kijas et al., 1998) different mutations in the MC1R gene have been associated with different coat colors. In cattle, the MC1R gene has been mapped to chromosome 18. It consists of a single exon of approximately 950 base pair (bp) and encodes for a protein of 45 kDa that belongs to the family of G protein-coupled receptor (Werth et al., 1996).
This protein, which contains seven transmembrane domains, is integrated in the cell membrane of melanocytes. It binds externally to the hormone MSH (melanocyte stimulating hormone) and to the product of the agouti gene (ASIP), to adjust the chain that leads to metabolic formation of eumelanin and pheomelanin (Mountjoy et al., 1992). Different alleles have been identified at MC1R locus in cattle. Three are the main ones (Klungland et al., 1995):
− allele “wild type” E+ that produces different colors (Adalsteinsson et al.,
1995);
− the dominant allele Ed (characterized by a point mutation that changes the
receptor and makes it constitutively expressed and determines the black color (Crepaldi et al., 2003);
− the e allele, characterized by a deletion which causes a shift in the reading of codons, inserts a stop codon, and gives rise to a non-functional protein. In homozygous condition causes the coat red/yellow color (Russo et al., 2007).
Apart from the above described three main alleles, the MC1R locus exhibits other polymorphisms whose effect on coat pigmentation are still is not well clarified. Among these, the E1 allele, the allele Ed1 and the ef allels can be mentioned.
The E1 allele is characterized by an insertion of 12 bp, which creates a duplication of amino acids (Gly, Ile, Ala, Arg) in position 224 of the amino acid sequence (Rouzaud et al., 2000; Maudet and Taberlet, 2002). The allele Ed1 is determined by a point mutation (C>T) in position 667 of the nucleotide sequence that causes an amino acid change (Arg>Trp) in position 223 of the amino acid sequence (Maudet and Taberlet, 2002; Graphodatskaya et al., 2002).
The ef allele, found only in few subjects in the Simmental breed. It is determined by a point mutation in position 890 to the nucleotide sequence (C>T), which causes a change in an amino acid (Thr>Ile) in position 297 of the protein sequence (Graphodatskaya et al., 2002).
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Melanocortin 1 receptor (MC1R) gene polymorphisms in three Italian cattle breeds
Anna Maria GuastellaA, Silvia SorboliniB, Antonio ZuccaroA, Elia PintusB, Salvatore BordonaroA, Donata MarlettaA,C and Nicolò Pietro Paolo MacciottaB
ADISPA Sezione di Scienze delle Produzioni Animali, Università degli Studi
di Catania, via Valdisavoia, 5. 95123 Catania, Italy.
BDipartimento di Scienze Zootecniche, Università degli Studi di Sassari, via
De Nicola, 9. 07100 Sassari, Italy.
CCorresponding author. Email: d.marletta@unict.it
This research has been pubished as:
A.M. Guastella, S. Sorbolini, A. Zuccaro, E. Pintus, S. Bordonaro, D. Marletta, and N.P.P. Macciotta. 2011. Melanocortin 1 receptor (MC1R) gene polymorphisms in three Italian cattle breeds. Animal Production Science, 2011, 51, 1039–1043.
Animal Production Science, 2011, 51, 1039–1043. Received 3 May 2011, accepted 8 September 2011, published online 21 October 2011.
Abstract
The Melanocortin 1 receptor (MC1R) is one of the main genes implicated in the determination of the coat colour in mammals. This locus showed a relevant genetic variation between breeds that can be exploited for breed traceability of the animal productions. Modicana, Cinisara and Sardo-Modicana are three Italian endangered cattle breeds. Genetic characterization by molecular markers is a fundamental prerequisite for managing genetic resources and for developing potential genetic traceability protocols. In order to improve the knowledge on Modicana, Cinisara and Sardo-Modicana breeds and to evaluate the possibility to develop DNA-based protocols for their mono-breeds products traceability, the genetic structure of MC1R gene was analysed. Four main alleles were observed in a representative sample of 162 animals. In the black coated Cinisara breed (n=42), the ED and E+ alleles segregated with a frequency of 0.93 for ED allele. In the red coated Modicana (n=60) and Sardo-Modicana (n=60) breeds the E+ and E1 alleles segregated with frequencies of 0.42, 0.57 and 0.52, 0.47, respectively. The recessive allele e showed a low frequency (0.01) in both breeds. Sequencing a subsample of 34 animals the rare E2 allele was found only in Modicana and Sardo-Modicana at a good frequency (0.50). A new PCR-RFLP test, based on BstOI restriction endonuclease, was devised to assay for this allele. Results of the work indicate that red coat in Modicana and Sardo-Modicana cattle is genetically determined by the E+ and E1 alleles instead of the e allele at homozygote status, as occurs in other red European breeds. In these three Italian breeds of local importance, MC1R polymorphisms can be used to discriminate Cinisara from
Modicana and Sardo-Modicana, but it was not able to distinguish between the two red coat populations.
Additional keywords: breed traceability, Cinisara, coat colour gene, genetic diversity, Modicana, Sardo-Modicana.
Introduction
Coat colour in mammals is determined by the distribution and relative amount of two pigments, eumelanin (black or brown pigment) and pheomelanin (red or yellow pigment) (Klungland and Våge, 2000). Melanin production is mainly regulated by two loci, namely Extension and Agouti (Seo et al., 2007). The Extension locus encodes for Melanocortin 1 Receptor (MC1R), a seven trans-membrane domain receptor. In cattle, the MC1R gene is located on chromosome 18 and consists of a single exon 954 bp long (Werth et al., 1996). This gene shows a polymorphism related to the coat colour (Olson, 1999). More recently, it has been proposed as breed-specific DNA marker for the genetic traceability of the animal productions (Chung et al., 2000; Maudet and Taberlet, 2002; Crepaldi et al., 2003; Rolando and Di Stasio, 2006).
Four main alleles responsible for coat colour determination have been identified at the MCR1 locus in cattle (Klungland et al., 1995; Joerg et al., 1996; Rouzaud et al., 2000; Kriegesmann et al., 2001; Maudet and Taberlet, 2002): (1) the wild-type E+, which may produce a wide range of colours, depending on genotype at the Agouti locus; (2) the dominant ED, that results in black coat; (3) the recessive e, which is associated with red/yellow coat colour in homozygotes; and (4) the E1 with an unclear role in colour determination (Crepaldi et al., 2005; Russo et al., 2007).
Furthermore a rare allele, now named E2, was previously observed in some Italian breeds (Maudet and Taberlet, 2002). The genetic polymorphism at MC1R gene has been investigated in several cattle breeds (Kantanen et al., 2000;
Rouzaud et al., 2000; Graphodatskaya et al., 2002; Maudet and Taberlet, 2002;Gan et al., 2007; Mohanty et al., 2008). This locus is a potential candidate marker for a genetic traceability test that could be used to certify typical livestock production. In Italy, for example, MC1R was found to be effective in distinguishing Parmigiano Reggiano cheese made from milk of the local breed Reggiana from other breeds as Holstein Friesian or Italian Brown (Russo et al., 2007). It may, therefore, also be used in other breeds of local importance as a population-specific marker.
An interesting situation is represented by three local cattle breeds farmed in extensive traditional systems in the two main Italian Islands. The Modicana (MO), characterised by a solid red coat, and the Cinisara (CI), characterised by a uniform black coat, are farmed in Sicily and their economic importance lies on the production of two typical “pasta filata” cheeses: Ragusano P.D.O. (Protected Designation of Origin) and “Caciocavallo Palermitano” cheese (Marletta et al., 1998; Guastella et al., 2006). The Sardo-Modicana (SM), derived by the cross of local Sarda cows with MO bulls (Dattilo and Brandano, 1969) is characterised by a wine red coat colour more intense in males. It is farmed extensively in Sardinia and the milk is used to produce the typical “Casizolu” cheese. More information about the breeds is available at the following link (http://eng.agraria.org/cattle. htm, verified 22 September 2011).
In the last 50 years these local breeds have experienced a progressive reduction in size, mainly due to the mechanization of agriculture and to the introduction of cosmopolitan breeds, more specialized and productive. European
Union policy supports their conservation, however they could definitely benefit from the creation of P.D.O. labels for their mono-breed products. An essential prerequisite for such an application is the knowledge of the genetic polymorphism of some candidate genes. In this paper, the genetic polymorphism of MC1R locus in MO, CI and SM cattle breeds was investigated to asses the feasibility of DNA-based traceability protocols for the identification of their mono-breed products.
Materials and methods Sampling
Blood samples were obtained from a representative sample of 162 cattle of the three breeds: 60 MO, 42 CI and 60 SM. Modicana was collected all over Sicily, CI mainly in the West of Sicily and SM in the Monti Ferru area of Middle-West Sardinia. Unrelated or minimally related individuals were chosen. Genomic DNA was extracted using the commercial GenElute Blood Genomic DNA kit (Sigma-Aldrich, St Louis, MO, USA).
Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction–amplified product length polymorphism (PCR-APLP) methods
The four main alleles (ED, E+, E1 and e) at the MC1R locus were determined by different protocols. A PCR-RFLP method, using MspI and MspaI1 restriction enzymes (New England BioLabs Inc., Milano, Italy), was used to identify E+, ED and e alleles (Rolando and Di Stasio, 2006). A PCR-APLP method was able to detect the 12-bp duplication that characterises the E1 allele (Russo et al., 2007). Amplifications were performed using a GenAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) thermal cycler. To resolve the presence of nucleotidic duplication, the PCR products were run on 5% polyacrylamide gel in a vertical apparatus (Sequi-Gen Sequencing Cell, BIO-RAD, Laboratories, Hercules, CA, USA).
DNA sequencing and PCR-RFLP method for detection of the E2 allele For sequence analysis, in order to confirm the insertion of 12 bp starting from position 669, a fragment was amplified in a subsample of 34 cows (9 MO, 4 CI and 21 SM) by using the following primers (forward 5'-TCG TGG AGA ACG TGC TGG TAG-3'; reverse 5'-TCC ACA ATG GCG TTG CAA ATG ATG-3') designed from the MC1R gene sequence (GenBank accession number Y19103). The PCR reaction was performed in a 25-μl mixture, containing 7–100 ng of genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 10 pmol of each
primer, 2 U of Ampli Taq DNA Gold Polymerase (Applied Biosystems). After 5 min of denaturation at 95°C, the PCR conditions were for 35 cycles at 95°C for 30 s, 62°C for 30 s, 72°C for 30 s and a final extension at 72°C for 10 min using a 2720 Thermal Cycler (Applied Biosystems). The amplified region ranged from positions 158 to 882 and contained all the known mutation sites. PCR products were resolved in 1.5% agarose gel, purified by Wizard Vs Gel and PCR Cleaning-up System (Promega Corporation, Madison, WI, USA) and sequenced using the BigDye Terminator Kit, on an ABI PRISM 3130 Genetic Analyser equipped with Sequencing Analysis software (Applied Biosystems).
The transition C667T that characterises the E2 allele creates an additional restriction site for BstOI (CCvTGG). A PCR-RFLP procedure for detection of the E2 allele was applied using the abovementioned primers and conditions. The amplicons were digested for 4 h at 60°C with 5 units of BstOI restriction enzyme (Promega, Carlsbad, CA, USA). Restriction fragments were separated on 4% MS-12 (Molecular Screen) agarose gels (PRONADISA, Torrejon de Ardoz,
Madrid, Spain) with GeneRule 50-bp DNA Ladder, stained with ethidium bromide and visualized under UV light.
